Circulating tumor cells (hereinafter; CTC) are rare tumor cells present in the blood and circulate through the body, and are known to be present at a rate of 1/1,0000,000 blood cells of a cancer patient. Despite the presence of only an extremely small amount in the blood, the presence of CTCs has been known as an important factor for the prognosis associated with survival rate in patients with metastatic breast cancer, colorectal cancer, and prostate cancer. Recently, there were reports that therapeutic responses can be easily predicted by monitoring tumor-specific markers of CTCs in the blood.
In the conventional diagnosis of cancer using blood, the prognosis of cancer occurrence was predicted based on the increase in expression of particular enzymes, etc., and Korean Patent Application Publication No. 2003-0036010 discloses a method for cancer diagnosis by measurement of enzyme activity of protein kinase in the blood.
However, until now, the detection of CTCs in the blood, which are not particular enzymes, has been mostly limited to the quantitative analysis of estimating the amount of tumor cells in the blood, i.e., the degree of tumor burden. If it is possible to collect the detected CTCs and analyze them pathologically, the method will be able to replace the performance of a histological examination for patients with terminal cancer, who have difficulty in receiving histological examination, reduce expenses for unnecessary examination, and indirectly reduce the cost for treatment by performing appropriate treatments suitable for the histological type of cancer, and thus the study has been continued.
Examples of the representative methods known so far by such study include an immunological assay using monoclonal antibodies and a separation method according to cell size, which are at the early stage of development and are partially commercialized. However, the method used at present is a test method for a one-time use, which only enables a simple count of CTCs or one or two times of immunocytochemical staining regarding the CTCs. Additionally, the immunological assay has a disadvantage in that, in the case of CTCs, hepatocellular carcinoma or malignant melanoma which exhibits an epithelial-mesenchymal transition (EMT), false-negatives may appear because the markers may not respond to cancer cells. Furthermore, the separation method according to cell size, which collects CTCs using a micro filter membrane with pores, has disadvantages in that the method, for lacking the variety in pore size, cannot filter the cancer cells which are smaller than the pores in the micro filter membrane, whereas the pore size may be clogged by the aggregation of white blood cells. Accordingly, both methods have fatal problems.
Additionally, the convention method of diagnosis was to extract only CTCs by removing red blood cells by chemical treatment followed by removing white blood cells by micro filtration method or surface markers, which required many steps before extracting the final CTCs, thus having a problem of reducing recovery rate of CTCs.
As a method of resolving the above problem, the CTCs may be detected using paraffin blocks which include circulating tumor cells.
Conventionally, paraffin blocks are being used to figure out the shapes of cells or tissues and, based on the results, use them for the diagnosis, treatment, and research of diseases via special staining, immunohistochemical staining, etc. The application of the paraffin blocks are generally performed using biopsy specimens. Upon fixation, the tissues go through with a process of washing, dehydration, clearing, and paraffin penetration to prepare paraffin blocks, which are then cut into a predetermined thickness and dried.
For the application of the paraffin blocks into blood cells, the blood cells should be fixed in a fixing solution to prepare cell blocks and then prepared into blood sections. In particular, examples of the conventional methods of preparing cell blocks for the biopsy specimens include: a) a fixed sediment method, in which centrifuged sediment is fixed in formalin; b) an egg-albumin method, which employs the principle that egg-albumin is agglutinated in ethanol; c) an agar method; d) a plasma thrombin clot method; e) collodion bag method; f) Millpore filter method, etc.
However, when an agar gel is used for the preparation of the cell blocks among the conventional methods, it has a problem in that the agar gel which supports the specimen, and thus significantly increase the time required for the preparation of paraffin blocks and the cost involved thereof thereby not being able to obtain satisfied results.
Additionally, when the fixed sediment method which precipitates specimens via centrifugation is used for the blood cells among the conventional methods, it has problems in that the amount of the cell block becomes smaller or the cells or tissues become spread due to the destruction and loss of the blood cells, and it becomes difficult to distinguish the cell block from paraffin after paraffin penetration, and it is impossible to embed using a forceps during the embedding process.
Additionally, when the paraffin block specimens prepared using the blood cells into cell blocks using the conventional methods are stained, it is difficult to perform a slide reading due to the background staining, and also there is a problem in that the sensitivity and specificity for the detection of targeted particular blood cells deteriorate due to the destruction and loss of the blood cells.
Furthermore, the subjects of diagnosis using the paraffin blocks are the conventional biopsy specimens or body cells, and these biopsy specimens or body cells have good intercellular adhesion and contain extracellular matrix components which aggregate cells, and thus were easy for preparing cell blocks. However, blood cells neither have the intercellular adhesion nor contain the extracellular matrix components which aggregate cells, and thus they are in a separate state without being aggregated. The blood cell sample in this state cannot allow the blood cells to aggregate, and thus regardless of the use of various methods for preparing cell blocks described above, the blood cells would not be aggregated. Accordingly, when paraffin blocks are prepared for the blood cells, the vanishing or loss of the blood cells during the preparation process may frequently occur thus making it difficult to prepare the intended paraffin blocks.
Even furthermore, when blood cells are subjected to the process of formalin fixation or paraffin block preparation without being properly aggregated, the blood cells themselves may be unprotected by being exposed to the outside, and thus the destruction, vanishing, or loss may occur more frequently. In particular, the CTCs, although included in the blood, are contained in an extremely small number. Therefore, when the blood cells being destroyed, vanished, or lost are the CTCs, it can cause a fatal problem in that CTCs cannot be diagnosed via the prepared paraffin blocks.